Treatment and prevention of helicobacter infection

ABSTRACT

An antigenic preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, comprises the catalase enzyme of Helicobacter bacteria, particularly the catalase enzyme of H. pylori or H. felis, or an immunogenic fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/AU95/00335, dated Jun. 8, 1995, and designating theUnited States of America, the disclosure of which is incorporated hereinby reference.

FIELD OF THE INVENTION

This invention relates to protective Helicobacter antigens, especiallyH. pylori antigens, and to the use of these antigens for the treatmentand prevention of gastroduodenal disease associated with H. pyloriinfection in humans.

BACKGROUND OF THE INVENTION

Helicobacter pylori is a bacterium that infects the stomach lining (orgastric mucosa) of perhaps half the world's population. Spiral organismswere first microscopically observed in human gastric mucosa in 1906.However, H. pylori was not successfully cultured until 1982. Infectionwith the organism is usually chronic, and results in continuinginflammation of the gastric mucosa. The infection is often asymptomatic.However, in association with other cofactors, a proportion of infectedpeople go on to develop sequelae including peptic ulceration of thestomach or duodenum, gastric adenocarcinomas and gastric lymphomas.Peptic ulcer treatment studies have shown that cure of H. pyloriinfection is associated with a dramatic reduction in the relapse rate ofthis usually chronic disease. Long term infection with H. pylori leadsto the development of chronic atrophic gastritis, which has long beenrecognised as a precursor lesion in the development of gastric cancer.Thus a number of studies have now linked preceding H. pylori infectionwith an increased risk of developing gastric cancer. Thereforeeradication of current infection and prevention of new infection withthis organism has the potential to significantly reduce the incidence ofdiseases that result in considerable morbidity and mortality¹,2.

Infection with H. pylori is difficult to treat. Current experimentaltherapies for treating the infection have problems with efficacy andsignificant levels of adverse effects. There are no prophylacticmeasures available. A solution to both the prevention and treatment ofH. pylori infection would be the development of an immunogenicpreparation that, as an immunotherapeutic, treated establishedinfections, and as a vaccine, prevented the establishment of new orrecurrent infections. Such a preparation would need to induce effectiveimmune responses to protective antigens, while avoiding inducingresponses to self antigens or other potentially harmful immuneresponses. This may be achieved by identifying the specific protectivecomponent or components and formulating immunotherapeutic or vaccinepreparations including these component(s).

The identification of such protective components of an organism, isoften accomplished through the use of an animal model of the infection.Initially, H. pylori did not naturally infect laboratory animals.However, an animal model of human H. pylori infection has been developedusing a closely related organism, H. felis, and specific pathogen free(SPF) mice³. These organisms are able to colonise the gastric mucosa ofSPF mice, where they establish a chronic infection with many of thefeatures of H. pylori infection in humans. H. felis infection in themice induces a chronic gastritis and a raised immune response. As in thehuman case, this response is not effective in curing the infection.

This model has been used to demonstrate that oral treatment of H. felisinfected mice with a preparation containing disrupted H. pylori cellsand cholera toxin as a mucosal adjuvant, can cure a significant portionof infected mice⁴. This effect is likely to be mediated through animmune response to a cross-reactive antigen possessed by each of theclosely related species.

In working by the inventors leading to the present invention, thesecross-reactive antigens were recognised by performing a Western blotusing H. pylori disrupted cells as the antigen, and probing the blotwith serum from mice immunised with H. felis and cholera toxin adjuvant.Sections of membrane containing proteins recognised as cross-reactivewere removed from the membrane, the proteins bound to them were eluted,and their N-terminal amino acid sequence determined by microsequencing.

The N-terminal amino acid sequence of one of the two proteins thatsuccessfully yielded sequence data closely matched the previouslypublished sequence of the microbial enzyme, urease⁵. This enzyme hasalready been shown to be a protective antigen when used in a vaccine toprevent infection.

The N-terminal amino acid sequence of the other protein closely matchedthe previously published N-terminal sequence of the microbial enzyme,catalase⁶. This enzyme has not previously been shown to be a protectiveantigen of H. pylori.

International Patent Application No. PCT/FR95/00383 (Publication No. WO95/27506) in the name Pasteur Merieux Serums et Vaccins, published Oct.19, 1995, discloses an H. pylori immunising composition, based onproposed use of H. pylori catalase in substantially purified form as animmunising substance useful for prophylactic or therapeutic purposes. Itis suggested that the catalase could be obtained either by extractionfrom H. pylori (using the purification method of Hazell et al.¹⁰) or byrecombinant means. The disclosure contains no supporting data showingefficacy of H. pylori catalase in use as an immunising substance, nor isthere any supporting disclosure or teaching of the preparation of H.pylori catalase by recombinant means or of the efficacy of recombinantcatalase in use as an immunising substance.

Recently, an H. pylori (Sydney strain)/mouse model of human H. pyloriinfection has been developed and used by the present inventors toconfirm that catalase, in particular recombinant catalase, has utilityas a protective antigen.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an antigenic preparationfor use in the treatment or prevention of Helicobacter infection, whichcomprises an at least partially purified preparation of the catalase ofHelicobacter bacteria.

The term "at least partially purified" as used herein denotes apreparation in which the catalase content is greater, preferably atleast 30% and more preferably at least 50% greater, than the catalasecontent of a whole cell sonicate of Helicobacter bacteria. Preferably,the preparation is one in which the catalase is "substantially pure",that is one in which the catalase content is at least 80%, morepreferably at least 90%, of the total Helicobacter antigens in thepreparation.

Accordingly, in a particularly preferred embodiment, the presentinvention provides an antigenic preparation for use in treatment orprevention of Helicobacter infection, which comprises substantially purecatalase of Helicobacter bacteria. Such a preparation may be prepared asa recombinant catalase by techniques described hereinafter.

In another aspect, the present invention provides an isolatedHelicobacter antigen for use in the treatment or prevention ofHelicobacter infection in a mammalian host, which comprises the catalaseof Helicobacter bacteria, or an immunogenic fragment thereof.

The term "isolated" as used herein denotes that the antigen hasundergone at least one purification or isolation step, and preferably isin a form suitable for use in a vaccine composition.

It is to be understood that the present invention extends not only to anantigenic preparation or isolated antigen comprising the catalase ofHelicobacter bacteria, but also to antigenic preparations comprisingimmunogenic fragments of this catalase, that is catalase fragments whichare capable of eliciting a specific protective immune response in amammalian host. Such immunogenic fragments may also be recognised byHelicobacter-specific antibodies, particularly monoclonal antibodieswhich have a protective or therapeutic effect in relation toHelicobacter infection or polyclonal antibodies contained in immune serafrom mammalian hosts which have been vaccinated against Helicobacterinfection.

In another aspect, the present invention provides a vaccine compositionfor use in the treatment or prevention of Helicobacter infection in amammalian host, which comprises an immunologically effective amount ofan antigenic preparation or isolated antigen as broadly described above,optionally in association with an adjuvant, together with one or morepharmaceutically acceptable carriers and/or diluents.

In yet another aspect, the present invention provides a method for thetreatment or prevention of Helicobacter infection in a mammalian host,which comprises administration to said host of an immunologicallyeffective amount of an antigenic preparation or isolated antigen asbroadly described above, optionally in association with an adjuvant.

In a related aspect, this invention provides the use of a vaccinecomposition comprising an immunologically effective amount of anantigenic preparation or isolated antigen as broadly described above,optionally in association with an adjuvant, for the treatment orprevention of Helicobacter infection in a mammalian host.

In yet another aspect, the invention provides the use of an antigenicpreparation or isolated antigen as broadly described above, optionallyin association with an adjuvant, in the manufacture of a vaccinecomposition for the treatment or prevention of Helicobacter infection ina mammalian host.

Preferably, but not essentially, the antigenic preparation or isolatedantigen of this invention is orally administered to the host, and isadministered in association with a mucosal adjuvant. However, theinvention also extends to parenteral administration of this antigenicpreparation or isolated antigen.

By use of the term "immunologically effective amount" herein in thecontext of treatment of Helicobacter infection, it is meant that theadministration of that amount to an individual infected host, either ina single dose or as part of a series, that is effective for treatment ofHelicobacter infection. By the use of the term "immunologicallyeffective amount" herein in the context of prevention of Helicobacterinfection, it is meant that the administration of that amount to anindividual host, either in a single dose or as part of a series, that iseffective to delay, inhibit or prevent Helicobacter infection. Theeffective amount varies depending upon the health and physical conditionof the individual to be treated, the taxonomic group of individual to betreated, the capacity of the individual's immune system to synthesiseantibodies, the degree of protection desired, the formulation of thevaccine, the assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

Preferably, the catalase antigen above comprises an amino acid sequencesubstantially corresponding to the deduced sequence of the catalase genefrom isolate RU1 or isolate 921023 hereinafter (SEQ ID NO.2 or 4), orallelic or other variants thereof. Suitable variants may have at least50-60%, more preferably at least 70-80%, and most preferably at least90%, similarity to one of the amino acid sequences referred to above, orto a region or part thereof, provided the variant is capable ofeliciting a specific protective immune response in a mammalian host.

As described above, the present invention extends not only to theparticular catalase antigen of Helicobacter bacteria as described above,but also to immunogenic fragments of the particular antigen, that isfragments of the antigen which are capable of eliciting a specificprotective immune response in a mammalian host. Suitably, theimmunogenic fragment will comprise at least five, and more preferably atleast ten, contiguous amino acid residues of the particular antigen.Such immunogenic fragments may also be recognised byHelicobacter-specific antibodies, particularly antibodies which have aprotective or therapeutic effect in relation to Helicobacter infection.

The present invention also extends to an antibody, which may be either amonoclonal or polyclonal antibody, specific for an antigenic preparationor an isolated Helicobacter antigen as broadly described above. Suchantibodies may be produced by methods which are well known to personsskilled in this field.

In this aspect, the invention further provides a method for thetreatment or prevention of Helicobacter infection in a mammalian host,which comprises passive immunisation of said host by administration ofan effective amount of an antibody, particularly a monoclonal antibody,specific for an antigenic preparation or an isolated Helicobacterantigen as broadly described above.

The Helicobacter antigenic preparation or isolated antigen of thisinvention may be prepared by purification or isolation from naturalsources, such as a whole cell sonicate of Helicobacter bacteria.Alternatively, however the antigenic preparation or isolated antigen maybe prepared by synthetic, preferably recombinant, techniques. Whenprepared by recombinant techniques, the antigen may have an amino acidsequence substantially identical to the naturally occurring sequence ormay contain one or more amino acid substitutions, deletions and/oradditions thereto provided that following such alterations to thesequence, the molecule is still capable of eliciting a specificprotective immune response against the naturally occurring Helicobacterantigen. A similar immunogenic requirement is necessary for anyfragments or derivatives of the antigen whether made from therecombinant molecule or the naturally occurring molecule. Accordingly,reference herein to a Helicobacter antigen is considered reference tothe naturally occurring molecule, its recombinant form and any mutants,derivatives, fragments, homologues or analogues thereof provided thatsuch molecules elicit a specific protective immune response against thenaturally occurring Helicobacter antigen. Also included are fusionmolecules between two or more Helicobacter antigens or with othermolecules including fusion molecules with other molecules such asglutathione-S-transferase (GST) or β-galactosidase.

The present invention also extends to an isolated nucleic acid moleculeencoding a Helicobacter catalase antigen and preferably having anucleotide sequence as set forth in SEQ ID NO. 1 or 3, or beingsubstantially similar to all or a part thereof. The term "substantiallysimilar" means having at least 40-50%, more preferably at least 60-70%,and most preferably at least 80% identity. A "part" in this contextmeans a continuous series of at least 15 nucleotides, and morepreferably at least 25 nucleotides.

According to this embodiment, there is provided a nucleic acid moleculecomprising a sequence of nucleotides which encodes a Helicobactercatalase antigen and hybridises under low stringency conditions to allor part of a nucleic acid sequence set forth in SEQ ID NO. 1 or 3, or toa complementary form thereof.

In another aspect, this invention provides a nucleic acid moleculecomprising a sequence of nucleotides substantially as set forth in SEQID NO. 1 or 3, or a part thereof.

The nucleic acid molecule may be RNA or DNA, single stranded or doublestranded, in linear or covalently closed circular form. For the purposesof defining the level of stringency, reference can conveniently be madeto Sambrook et al. (1989) at pp 387-389 which is herein incorporated byreference where the washing step at paragraph 11 is considered highstringency. A low stringency is defined herein as being in 0.1-0.5 w/vSDS at 37-45° C. for 2-3 hours. Depending on the source andconcentration of nucleic acid involved in the hybridisation, alternativeconditions of stringency may be employed such as medium stringentconditions which are considered herein to be 0.25-0.5% w/v SDS at ≧45°C. for 2-3 hours or high stringent conditions as disclosed by Sambrooket al. (1989).

It will be appreciated that the sequence of nucleotides of this aspectof the invention may be obtained from natural, synthetic orsemi-synthetic sources; furthermore, this nucleotide sequence may be anaturally-occurring sequence, or it may be related by mutation,including single or multiple base substitutions, deletions, insertionsand inversions, to such a naturally-occurring sequence, provided alwaysthat the nucleic acid molecule comprising such a sequence is capable ofbeing expressed as a Helicobacter antigen as broadly described above.

The nucleotide sequence may have expression control sequences positionedadjacent to it, such control sequences usually being derived from aheterologous source.

This invention also provides a recombinant DNA molecule comprising anexpression control sequence having promoter sequences and initiatorsequences and a nucleotide sequence which codes for a Helicobactercatalase antigen, the nucleotide sequence being located 3' to thepromoter and initiator sequences. In yet another aspect, the inventionprovides a recombinant DNA cloning vehicle capable of expressing aHelicobacter catalase antigen comprising an expression control sequencehaving promoter sequences and initiator sequences, and a nucleotidesequence which codes for a Helicobacter catalase antigen, the nucleotidesequence being located 3' to the promoter and initiator sequences. In afurther aspect, there is provided a host cell containing a recombinantDNA cloning vehicle and/or a recombinant DNA molecule as describedabove.

Suitable expression control sequences and host cell/cloning vehiclecombinations are well known in the art, and are described by way ofexample, in Sambrook et al. (1989).

In yet further aspects, there is provided fused polypeptides comprisinga Helicobacter catalase antigen of this invention and an additionalpolypeptide, for example a polypeptide coded for by the DNA of a cloningvehicle, fused thereto. Such a fused polypeptide can be produced by ahost cell transformed or infected with a recombinant DNA cloning vehicleas described above, and it can be subsequently isolated from the hostcell to provide the fused polypeptide substantially free of other hostcell proteins.

The present invention also extends to synthetic polypeptides displayingthe antigenicity of a Helicobacter catalase antigen of this invention.As used herein, the term "synthetic" means that the polypeptides havebeen produced by chemical or biological means, such as by means ofchemical synthesis or by recombinant DNA techniques leading tobiological synthesis. Such polypeptides can, of course, be obtained bycleavage of a fused polypeptide as described above and separation of thedesired polypeptide from the additional polypeptide coded for by the DNAof the cloning vehicle by methods well known in the art. Alternatively,once the amino acid sequence of the desired polypeptide has beenestablished, for example, by determination of the nucleotide sequencecoding for the desired polypeptide, the polypeptide may be producedsynthetically, for example by the well-known Merrifield solid-phasesynthesis procedure.

Once recombinant DNA cloning vehicles and/or host cells expressing aHelicobacter catalase antigen of this invention have been identified,the expressed polypeptides synthesised by the host cells, for example,as a fusion protein, can be isolated substantially free of contaminatinghost cell components by techniques well known to those skilled in theart.

Isolated polypeptides comprising, or containing in part, amino acidsequences corresponding to a Helicobacter catalase antigen may be usedto raise polyclonal antisera by immunising rabbits, mice or otheranimals using well established procedures. Alternatively, suchpolypeptides may be used in the preparation of monoclonal antibodies bytechniques well known in the art.

In addition, the polypeptides in accordance with this inventionincluding fused polypeptides may be used as an active immunogen in thepreparation of single or multivalent vaccines by methods well known inthe art of vaccine manufacture for use in the treatment or prevention ofHelicobacter infection in a mammalian host.

Alternatively, the polypeptides in accordance with the present inventionincluding fused polypeptides may be used as antigen in a diagnosticimmunoassay for detection of antibodies to Helicobacter in a sample, forexample, a serum sample from a human or other mammalian patient. Suchimmunoassays are well known in the art, and include assays such asradioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).

The present invention also extends to delivery to the host using avector expressing the catalase of Helicobacter bacteria, or animmunogenic fragment thereof. Accordingly, in a further aspect thisinvention provides a preparation for use in the treatment or preventionof Helicobacter infection in a mammalian host, which comprises a vectorexpressing the catalase of Helicobacter bacteria or an immunogenicfragment thereof.

In this aspect, the invention extends to a method for the treatment orprevention of Helicobacter infection in a mammalian host, whichcomprises administration to said host of a vector expressing thecatalase of Helicobacter bacteria or an immunogenic fragment thereof.

Further, the invention extends to the use of a vector expressing thecatalase of Helicobacter bacteria or an immunogenic fragment thereof,for the treatment or prevention of Helicobacter infection in a mammalianhost.

Throughout this specification, unless the context requires otherwise,the word "comprise", or variations such as "comprises" or "comprising",is to be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, the antigenic preparation or isolated antigen of thisinvention comprises the catalase of H. pylori or H. felis, mostpreferably H. pylori catalase. Preferably also, this antigenicpreparation or isolated antigen is used in a vaccine composition fororal administration which includes a mucosal adjuvant.

In a particularly preferred aspect of this invention, an oral vaccinecomposition comprising substantially pure H. pylori catalase, morepreferably recombinant H. pylori catalase, in association with a mucosaladjuvant is used for the treatment or prevention of H. pylori infectionin a human host.

The mucosal adjuvant which is optionally, and preferably, administeredwith the Helicobacter catalase preparation or antigen to the infectedhost is preferably cholera toxin. Mucosal adjuvants other than choleratoxin which may be used in accordance with the present invention includenon-toxic derivatives of cholera toxin, such as the B sub-unit (CTB),chemically modified cholera toxin, or related proteins produced bymodification of the cholera toxin amino acid sequence. These may beadded to, or conjugated with, the Helicobacter catalase preparation orantigen. The same techniques can be applied to other molecules withmucosal adjuvant or delivery properties such as Escherichia coli heatlabile toxin. Other compounds with mucosal adjuvant or delivery activitymay be used such as bile; polycations such as DEAE-dextran andpolyornithine; detergents such as sodium dodecyl benzene sulphate;lipid-conjugated materials; antibiotics such as streptomycin; vitamin A;and other compounds that alter the structural or functional integrity ofmucosal surfaces. Other mucosally active compounds include derivativesof microbial structures such as MDP; acridine and cimetidine.

The Helicobacter catalase preparation or antigen may be delivered inaccordance with this invention in ISCOMS (immune stimulating complexes),ISCOMS containing CTB, liposomes or encapsulated in compounds such asacrylates or poly(DL-lactide-co-glycoside) to form microspheres of asize suited to adsorption by M cells. Alternatively, micro ornanoparticles may be covalently attached to molecules such as vitaminB12 which have specific gut receptors. The Helicobacter catalasepreparation or antigen may also be incorporated into oily emulsions anddelivered orally. An extensive though not exhaustive list of adjuvantscan be found in Cox and Coulter⁷.

Other adjuvants, as well as conventional pharmaceutically acceptablecarriers, excipients, buffers or diluents, may also be included in theprophylactic or therapeutic vaccine composition of this invention. Thevaccine composition may, for example, be formulated in enteric coatedgelatine capsules including sodium bicarbonate buffers together with theHelicobacter catalase preparation or antigen and cholera toxin mucosaladjuvant.

The formulation of such therapeutic compositions is well known topersons skilled in this field. Suitable pharmaceutically acceptablecarriers and/or diluents include any and all conventional solvents,dispersion media, fillers, solid carriers, aqueous solutions, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and it isdescribed, by way of example, in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activeingredient, use thereof in the pharmaceutical compositions of thepresent invention is contemplated. Supplementary active ingredients canalso be incorporated into the compositions.

As an alternative to the delivery of the Helicobacter catalasepreparation or antigen in the form of a therapeutic or prophylactic oralvaccine composition, the catalase or an immunogenic fragment thereof maybe delivered to the host using a live vaccine vector, in particularusing live recombinant bacteria, viruses or other live agents,containing the genetic material necessary for the expression of thecatalase or immunogenic fragment as a foreign antigen. Particularly,bacteria that colonise the gastrointestinal tract, such as Salmonella,Yersinia, Vibrio, Escherichia and BCG have been developed as vaccinevectors, and these and other examples are discussed by Holmgren et al.⁸and McGhee et al.⁹.

The Helicobacter catalase preparation or antigen of the presentinvention may be administered as the sole active immunogen in a vaccinecomposition or expressed by a live vector. Alternatively, however, thevaccine composition may include or the live vector may express otheractive immunogens, including other Helicobacter antigens such as ureaseor the lipopolysaccharide (LPS) of Helicobacter bacteria (seeInternational Patent Application No. PCT/AU95/00077), as well asimmunologically active antigens against other pathogenic species.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the human subjects to be treated; each unitcontaining a predetermined quantity of active ingredient calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier and/or diluent. The specifications for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active ingredient andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active ingredientfor the particular treatment.

Data obtained from Western blots mentioned above, show that H. pyloricatalase is recognised by the serum of mice vaccinated with an H. felisantigen preparation (plus cholera toxin adjuvant). These mice can beshown to be protected against H. felis infection. This data indicatesthe use of H. pylori catalase as a protective antigen in human H. pyloriinfection, and purified or recombinant catalase may be used as anantigenic component of a therapeutic or prophylactic vaccine, either onits own, or in combination with other antigens, carriers, adjuvants,delivery vehicles or excipients.

Further details of the present invention are set out, by way ofillustration only, in the following Examples. It is to be understood,however, that this detailed description is included solely for thepurposes of exemplifying the present invention, and should not beunderstood in any way as a restriction on the broad description of theinvention as set out above.

EXAMPLE 1

A. METHODS

Sonicated H. pylori cells (strain HP 921023) were separated in a 12%discontinuous (i.e. homogeneous) SDS-PAGE gel under denaturingconditions using a Mini-Protean II apparatus (Bio-Rad). Proteins weretransferred from the gel to ProBlott (Applied BiosciencesPVDF-polyvinylidene difluoride) membrane using CAPS buffer(3-(cyclohexylamino)-1-proanesulphonic acid buffer) in a Mini transblotsystem (Bio-Rad).

Strips were removed from the ends of the PVDF and reacted with immunesera from mice vaccinated with H. felis plus cholera toxin and tracedwith an HRP labelled anti-mouse sera and developed using4-chloro-1-naphthol as per standard Western blot methods. The remainderof the PVDF was stained with Coomassie blue (Bio-Rad) to visualise theprotein bands. Six proteins recognised by the immune sera were selectedand the corresponding Coomassie stained bands on the PVDF were carefullyexcised for sequencing.

The six excised bands of PVDF were cut into small pieces (approx. 0.5 cmlong) and placed into the reaction cartridge of an Applied BiosystemsModel 476A Protein Sequencer System. All chemistry, HPLC separations,data quantitation and protein sequencing reporting is automaticallycarried out in this system.

B. RESULTS

Four samples gave no signal in the Protein Sequencer System. Two samplesgave clear amino acid sequence data: sample 5, an approximately 53 kDprotein (±10%), and sample 3, an approximately 66 kD protein (±10%).This data is shown below.

(i) Sample 3:

    ______________________________________                                        D D N                                                                          M K K I V F K E Y V (SEQ ID NO:5)                                             A P                                                                          ______________________________________                                    

Note: the first three cycles gave equivocal results.

The sequence data of sample 3 corresponds closely, but not exactly, withthe previously published N-terminal sequence for the enzyme urease⁵.This enzyme has been shown to be a protective antigen in studies usingthe H. felis/mouse model.

(ii) Sample 5:

    M V N K D V K Q T T A F G T P (SEQ ID NO:6)

The sequence data of sample 5 corresponds closely, with one difference,to the previously published N-terminal sequence of the enzyme catalase⁶.This enzyme has not previously been shown to be a protective antigenhowever the fact that the enzyme is recognised by the immune serum ofmice vaccinated with an H. felis antigen preparation to protect againstH. felis infection, combined with the fact that mice vaccinated with anH. pylori antigen preparation are protected against H. felis infection,indicates the H. pylori catalase as a protective antigen in H. pyloriinfection in humans.

EXAMPLE 2

1. PURIFICATION OF H. PYLORI CATALASE¹⁰

Approximately 60 plates (CSA) of H. pylori (clinical strain 921023) weregrown in 10% CO₂ at 37° C. for 48 hours. All following steps untilloading on the column were undertaken on ice. The H. pylori cells wereharvested in 0.1 M sodium phosphate buffer pH 7.2 and the suspensionspun down gently and resuspended in no more than 5 mL of 0.1 M sodiumphosphate buffer. The suspension was then sonicated at 6 kHz 40% dutycycle for 5 minutes. Following this, the sonicate was spun for 5 minutesat 10,000 g, the supernatant collected and passed through a 0.22 μmfilter into a sterile container.

The filtrate was loaded onto a K26/100 gel filtration column ofSephacryl S-300 HR and eluted using sodium phosphate buffer at a flowrate of 1.0 mL min⁻¹. The eluate was collected into fractions (100drops/fraction) and those containing catalase identified by testing forcatalase activity (1 drop of the fraction placed in H₂ O₂ diluted 1:10in distilled water and examined for bubbling). Fractions containing thestrongest catalase activity were pooled then diluted 1:10 in 0.01Msodium phosphate (filtered). The fractions were then run through aMEMSEP 1000 cm ion exchange capsule. 100 mL of the 0.01 M sodiumphosphate buffer was then run through the ion exchange capsule to removeany excess proteins. 1 M NaCl in 0.1 M sodium phosphate buffer was runthrough the ion exchange capsule to elute out the catalase. Catalasepositive fractions were identified by their strong yellow colour andconfirmed by testing for a bubbling reaction in H₂ O₂.

The catalase positive fractions were stored at 4° C. and protected fromlight. Each fraction was tested for protein concentration using theBio-Rad DC protein assay, and selected for immunising mice if itcontained over 1.5 mg/mL of protein. Prior to immunising mice thepurified catalase was checked for contaminants using 12% SDS-PAGE.Proteins were visualised by staining with Coomassie Blue, whichindicated that the catalase preparation was at least 95% pure. Imageanalysis indicated that the catalase's molecular weight was 52-53 kDa.The purified catalase was also strongly recognised by a catalasemonoclonal antibody.

2. IMMUNISATION WITH H. PYLORI CATALASE.

Sufficient purified catalase for immunising 10 mice was obtained andpooled. Mice were given 0.2 mg purified catalase +10 μg cholera toxin(CT) 4 times on days 0, 7, 14 and 21. Control groups were given choleratoxin alone or PBS buffer alone. The dose size was 150 μl for allgroups. On the day of each immunising dose, the catalase was checked foractivity using ₂ and for any signs of degradation using SDS-PAGE andCoomassie Blue staining. No signs of declining activity or anydegradation was observed throughout the immunisation course. Three weeksafter the last immunising dose all groups were challenged twice with˜10⁸ H. felis. Three weeks later mice were euthanased and samples (sera,saliva, bile and the stomach--half for histology and half the antrum forthe direct urease test) were collected.

    ______________________________________                                        Experiment Outline                                                                         CATALASE CT (10                                                                            CT ALONE  PBS ALONE                                   TIME (days) Mice) (10 mice) (10 mice)                                       ______________________________________                                         0       Cat + CT     CT alone    PBS alone                                      7 Cat + CT CT alone PBS alone                                                14 CAT + CT CT alone PBS alone                                                21 Cat + CT CT alone PBS alone                                                42 H. felis H. felis Challenge H. felis                                        Challenge  Challenge                                                         44 H. felis Challenge H. felis Challenge H. felis                                Challenge                                                                  65 Collect 10 Collect 10 Collect 10                                         ______________________________________                                    

3. RESULTS

    ______________________________________                                        Urease                                                                          POSITIVE UREASE RESULT (%)                                                    Catalase + CT     CT alone PBS alone                                          (10) (10) (10)                                                              ______________________________________                                        0/10 (0)        7/10 (70)                                                                              10/10 (100)                                          ______________________________________                                    

Western Blotting

Western blots of sera from mice showed strong recognition of H. pyloricatalase by the immunised mice, whereas mice from the other groupsshowed weak or absent recognition.

Persons skilled in this art will appreciate that variations andmodifications may be made to the invention as broadly described herein,other than those specifically described without departing from thespirit and scope of the invention. It is to be understood that thisinvention extends to include all such variations and modifications.

EXAMPLE 3

CLONING PURIFICATION AND TESTING IN AN H. PYLORI ANIMAL MODEL OFRECOMBINANT H. PYLORI CATALASE AS A PROTECTIVE ANTIGEN

The catalase gene has been cloned from each of two different isolates ofH. pylori, isolate RU1 and isolate 921023.

1. IDENTIFICATION OF E. COLI CLONES EXPRESSING CATALASE FROM H. PYLORIISOLATE 921023.

1.1 MATERIALS AND METHODS

1.1.1 Bacterial Strains

Helicobacter pylori strain HP921 023 was used as the DNA donor forpreparing the gene library. Escherichia coli strain ER1793 (New EnglandBiolabs) was the host used for phage infection and plating of Lambda ZAPExpress. E. coli strains XL 1-Blue MRF' and XLOLR (Stratagene) were usedfor excision of phagemid pBK-CMV and protein expression of the clonedgene.

1.1.2 Isolation of H. pylori Chromosomal DNA

Whole cell DNA from H. pylori was prepared essentially as reported byMajewski and Goodwin¹¹.

1.1.3 Antisera Preparation

Mouse antisera was raised against Helicobacter pylori by fourora-gastric immunisations at weekly intervals. Each vaccine doseconsisted of 1 mg (protein) of sonicated H. pylori and 10 μg of choleratoxin. Blood was collected and serum pooled. This serum was absorbedwith 50% v/v E. coli extract (Promega) containing 5% w/v skim milk and0.05% v/v Tween 20 in TBS at a final dilution of 1:100. The preparationwas incubated at room temperature for 4 hours prior to immunoscreeningto eliminate non-specific reactivity of antisera with host proteins. Thespecificity of the sera was confirmed by dot blot and Western blotting,using dilutions of whole cells of H. pylori for positive control and E.coli XLOLR as the negative control.

1.1.4 Bacterial Growth Conditions

For infection with Lambda ZAP Express, strain ER1793 cells wereinitially grown in Luria-Bertani (LB) broth supplemented with 0.2% w/vmaltose and 10 mM MgSO₄ at 20° C. Following infection, cells weremaintained in LB broth at 37° C. for 15 minutes and then plated on NZYagar medium and incubated at 42° C. for 4 hours then at 37° C.overnight. For phagemid excision and plasmid isolation E. coli strainsXL1-Blue and XLOLR were grown in LB broth at 37° C., and transformedXLOLR cells selected on LB/Kanamycin plates (50 μg/mL) at 37° C.

1.1.5 Construction of H. pylori Gene Library

An H. pylori expression library was constructed using standardprocedures¹², in the Lambda ZAP express vector (Stratagene) which hadbeen predigested with BamHI and the terminal 5' phosphates removed withcalf intestinal phosphatase. Genomic DNA partially digested with Sau3AI,was fractionated by gel electrophoresis and DNA fragments between 6 to12 kb were isolated. This DNA was ligated with 1.0 μg of BamHI-digestedlambda arms. Recombinant phage DNA was packaged in vitro using CigapackII extracts (Stratagene). The library was titred by infecting E. colistrain ER 1793 or XL1-Blue MRF' cells with aliquots of packaged phageand plated onto indicator plates containing IPTG and X-gal. The ratio ofnon-recombinant phage to recombinant phage was 1:5. The titre of therecombinant library was calculated to be 1×10⁶ pfu per μg of lambda DNA.

1.1.6 Screening of H. pylori Library for the Gene Coding for Catalase

A portion of the H. pylori gene library was screened by DNAhybridization techniques using a cloned probe comprising approximately200 bp from the H. pylori catalase coding sequence beginning atnucleotide 410. A total of 8000 plaques were plated (4000 bacteriophageplaques per plate) and lifted onto nitrocellulose filters for DNAhybridization analysis with a P-labelled probe. When a positive phageclone was identified, an agar plug containing the plaque was picked andphage eluted into SM buffer. To obtain plaque purity the processes ofinfecting bacteria, replating and hybridization were repeated.

1.1.7 in vivo Excision of Plasmid pBK-CMV Lambda ZAP Express Vector

In vivo excision of pBK-CMV containing H. pylori DNA from lambda ZAPExpress was achieved by infecting E. coli strain XL1-Blue MRF'simultaneously with Lambda ZAP Express containing insert DNA andExAssist helper phage M13. Excised phagemids were packaged asfilamentous phage particles and secreted from host cells, which weresubsequently heat killed. The phagemids were rescued by infecting XLOLRcells and plating onto LB/Kanamycin (50 μg/mL) plates. Bacterialcolonies appearing on plates contained pBK-CMV double-stranded phagemidwith the cloned DNA insert from H. pylori. These colonies were thenanalysed for catalase expression.

1.1.8 SDS-PAGE and Western Blot Analysis

The proteins produced by these potentially H. pylori catalase clones inE. coli XLOLR were analysed by standard SDS-PAGE and Western Blottechniques¹²,13. 10 mL cultures of XLOLR containing expression plasmidwere grown in supermedium at 37° C. overnight. Cultures were inducedwith IPTC to a final concentration of 1 mM, with continued incubationfor 2-4 h. Aliquots of 1 mL were collected, cells pelleted bycentrifugation and resuspended in 10 mM Tris-HCl (pH 8). Cells weremixed with equal volume of SDS sample reducing buffer and boiled for 10minutes. Proteins were resolved by electrophoresis on 4-20% gradientTris-glycine gels (Novex) and electrotransferred onto nitrocellulosemembrane (BioRad) for detection of immunoreactive proteins of H. pyloriusing anti-H. pylori mouse sera as described above.

1.1.9 Catalase Activity Assay

Colonies of candidate clones were grown on LB/Kanamycin (50 μg/mL)plates following which 100 μl of hydrogen peroxide was applied to eachcolony. A positive result was indicated by a characteristic bubbling ofthe cells due to the degradation of substrate and the release of oxygen.

1.1.10 DNA Sequencing

DNA sequence analysis was performed by manual sequencing on both strandsof plasmid DNA by primer walk using the dideoxynecleotide chaintermination method¹⁴.

1.1.11 Plasmid DNA Preparation

Plasmid DNA was isolated by the alkaline lysis method¹² from cultures ofE. coli XLOLR clones. Restriction enzyme digestion were performed asrecommended by the enzyme manufacturer (Promega Inc.).

1.2 RESULTS AND DISCUSSION

To enable isolation of clones bearing the complete Helicobacter pyloricatalase gene, a genomic library of strain HP921023 was constructed inthe lambda expression vector lambda ZAP Express. This library wasscreened with a radioactively labelled probe representing around 200 bpof the catalase sequence. Approximately 8000 plaques were screenedresulting in the detection of eight positive clones. These were picked,purified and the expression plasmid pBK-CMV excised for furthercharacterisation. The proteins expressed by the recombinant plasmidswere analysed by Western blotting. Of the eight clones tested, fivepossessed an extremely immunoreactive band at approximately 50,000 Da(the size expected for catalase).

To investigate whether this immunoreactivity correlated with biologicalactivity, bacterial colonies representing a positive and a negativereaction were tested for catalase activity by the addition of hydrogenperoxide. The immunoreactive clone exhibited an explosive bubblingaction whilst the non-immunoreactive clone remained unchanged.

From this data it was assumed that the positive clone contained thecomplete coding sequence for catalase and this clone was furthercharacterised by restriction mapping and DNA sequencing.

To enable localisation of the catalase coding sequence, the DNA sequenceof the 200 bp probe fragment was obtained and useful restrictions siteswere identified for mapping (HindII, BstXI, PflMI). From this data boththe direction and approximate position of the gene could be deduced.

DNA sequence analysis confirmed the mapping data and the catalasesequence coding for a protein of predicted molecular weight of 58,650 Dais shown in SEQ ID NO. 1 and 2.

2. IDENTIFICATION OF E. COLI CLONES EXPRESSING CATALASE FROM H. PYLORIISOLATE RU1.

2.1 MATERIALS AND METHODS

To obtain the catalase gene from H. pylori isolate RU1, genomic DNA fromH. pylori isolate RU1 was partially digested with Sau3A and cloned intothe λ-ZAP Express vector (Stratagene). This genomic library was probedwith a 711 bp fragment of the H. pylori catalase ORF, which wasgenerated by PCR using primers starting at nucleotide 1 (21 mer) andterminating at nucleotide 711 (18 mer), and labelled withdioxigenin-dUTP (Boehringer Mannheim).

Catalase positive clones were excised into phagemid form and introducedinto the XLOLR E. coli strain (Stratagene). Clones which produced afunctional catalase were selected by placing the cells into 30% H₂ O₂and checking for the rapid formation of oxygen. The selected strain wasmaintained on Luria agar containing 50 mg/L Kanamycin sulfate (CibcoBRL).

For recombinant catalase purification, the recombinant E. coli strainwas grown in Luria broth plus Kanamycin, and purified using the methodof Hazell et al¹⁰

DNA sequence analysis was performed by manual sequencing on both strandsof plasmid DNA by primer walk using the deoxynucleotide chaintermination method¹⁴.

2.2 RESULTS

DNA sequence analysis identified a sequence coding for a protein ofpredicted molecular weight of 58,650 Da, which is shown in SEQ ID NO. 3and 4.

3. PROTECTIVE EFFICACY OF RECOMBINANT H. PYLORI CATALASE FROM ISOLATERU1

3.1 MATERIALS AND METHODS

3.1.1 Immunisation with Recombinant H. pylori Catalase

Sufficient purified catalase (from isolate RU1) for immunising 10 micewas pooled. Female SPS BALB/c mice were given 0.2 mg purified catalase+10 μg cholera toxin (CT) 4 times on days 0, 7, 14 and 21.

Control groups were: H. pylori whole cell sonicate+CT (positivecontrol), E. coli and PBS buffer alone (negative controls). The dosesize was 150 μl for mice dosed with catalase and 100 μl for thoseanimals receiving other preparations. On the day of each immunisingdose, the catalase was checked for activity using H₂ O₂ and for anysigns of degradation using SDS-PAGE and Coomassie Blue staining. Nosigns of declining activity or any degradation was observed throughoutthe immunisation course. Three weeks after the last immunising dose thegroups were challenged with two doses of ˜10⁸ H. pylori (Sydney strain),given 48 hours apart. Three weeks later mice were euthanased and samples(sera, saliva, bile and the stomach--half for histology and half theantrum for the direct urease tests³) were collected. The histologysamples were fixed in 10% buffered formalin, paraffin embedded andstained using the May-Grunwald Giemsa stain. Stomach sections werescanned for H. pylori using light microscopy (1000× magnification) andscored as infected if one or more organisms were detected in either thegastric body or antrum.

3.1.2 Experimental Outline

    ______________________________________                                              rCat + CT                                                                              HP + CT  E. coli + CT                                                                          PBS Alone                                                                             Norm                                    Day [n = 10] [n = 10] [n = 10] [n = 10] [n = 10]                            ______________________________________                                         0    rCat + CT                                                                              Hp + CT  E.coli + CT                                                                           PBS Alone                                                                             --                                       7 rCat + CT Hp + CT E.coli + CT PBS Alone --                                 14 rCat + CT Hp + CT E.coli + CT PBS Alone --                                 21 rCat + CT Hp + CT E.coli + CT PBS Alone --                               42    H. pylori Challenge       --                                              44 H. pylori Challenge --                                                     58 Collect all groups Collect                                               ______________________________________                                    

3.2 RESULTS AND DISCUSSION

Infection was assessed by testing samples of antral mucosa in the rapidmicrotitre urease test as described in Lee et al.³ and by directmicroscopic examination. The urease test has been validated as beinghighly predictive of Helicobacter infection. Urease positivity indicatesHelicobacter infection. Mice were scored as infected if positive ineither test.

    ______________________________________                                        GROUP           No. Infected (%)                                              ______________________________________                                        Catalase + CT   1/10 (10)                                                       XLOLR + CT 9/10 (90)                                                          PBS Alone 10/10 (80)                                                          H. pylori + CT 2/10 (10)                                                      Normal (unchallenged) 0/8 (0)                                               ______________________________________                                    

This experiment indicates that recombinant H. pylori catalase is aneffective protective antigen for immunisation against H. pyloriinfection.

Previous results with sonicates of whole helicobacter cells⁴ and theresults of Corthesy-Theulaz et al.¹⁵ with a recombinant helicobacterantigen, urease, show that antigens that have a protective, orprophylactic effect to prevent new infections, also can be usedtherapeutically to treat current infections. Therefore it is expectedthat catalase could be used both as a protective antigen in a vaccine toprevent infection, and in an immunotherapeutic composition ortherapeutic vaccine to treat infected persons.

REFERENCES

1. Helicobacter pylori Biology and Clinical Practice (1993), Edited byC. Stewart Goodwin and Bryan W. Worsley. Published by CRC Press.

2. Halter, F., Hurlimann, S. and Inauen, W. (1992). Pathophysiology andclinical relevance of Helicobacter pylori. The Yale Journal of Biologyand Medicine, 65:625-638,

3. Lee, A., Fox, J. G., Otto, G. and Murphy, J. (1990). A small animalmodel of human Helicobacter pylori active chronic gastritis.Gastroenterology, 99:1316-1323.

4. Doidge, C. G., Gust, I., Lee, A., Buck, F., Hazel, S. and Mane, U.(1994). Therapeutic immunisation against Helicobacter pylori--The firstevidence. Lancet 343(i):914-915.

5. Clayton, C. L., Pallen, M. J., Kleanthous, H., Wren, B. W. andTabaqchali, S. (1990). Nucleotide sequence of two genes fromHelicobacter pylori encoding for urease subunits. Nucleic Acid Res.,18(2):362

6. Westblom, T. U., Phadnis, S., Langenberg, W., Yoneda, K., Madan, E.and Midkiff, B. R. (1992). Catalase negative mutants of Helicobacterpylori. European Journal of Clinical Microbiology and InfectiousDiseases, 11:522-526.

7. Cox, J. and Coulter, A. (1992). Advances in adjuvant technology andapplication. In Animal Parasite Control Using Biotechnology. Edited byW. K. Yong. Published by CRC Press.

8. Holmgren, J., Czerkinsky, C., Lycke, N. and Svennerholm, A-M. (1992).Mucosal Immunity: Implications for Vaccine Development. Immunobiol.184:157-179.

9. McGhee, J. R., Mestecky, J., Dertzbaugh, M. T., Eldridge, J. H.,Hirasawa, M. and Kiyono, H. (1992). The mucosal immune system: fromfundamental concepts to vaccine development. Vaccine 10(2):75-88.

10. Hazell, S. L., Evans Jr., D. J. and Graham, D. Y (1991).Helicobacter pylori catalase. J. Gen. Microbiol. 137:57-61.

11. Majewski, S. L. H., and Goodwin, C. S. (1988). Restrictionendonuclease analysis of the genome of Campylobacter pylori with a rapidextraction method: evidence for considerable genomic variation. J. Inf.Dis. 157(3):465-471.

12. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). MolecularCloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press.

13. Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. Proc. Natl. Acad. Sci USA 74:4350-4354.

14. Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

15. Corthesy-Theulaz, I, Porta, N., Glauser, M., Saraga, E., Vaney,A-C., Haas, R., Kraehenbuhl, J-P., Blum, A. L. and Michetti, P. (1995).Oral Immunisation With Helicobacter pylori Urease B Subunit as aTreatment Against Helicobacter infection in Mice. Gastroenterology109:115-121.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 6                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1518 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Heliobacter - #pylori                                           (B) STRAIN: HP 921023                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1515                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 1..1515                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG GTT AAT AAA GAT GTG AAA CAA ACC ACT GC - #T TTT GGC ACT CCC        GTT       48                                                                    Met Val Asn Lys Asp Val Lys Gln Thr Thr Al - #a Phe Gly Thr Pro Val            1               5 - #                 10 - #                 15              - - TGG GAT GAC AAC AAT GTG ATT ACG GCC GGC CC - #T AGA GGT CCT GTT TTA           96                                                                       Trp Asp Asp Asn Asn Val Ile Thr Ala Gly Pr - #o Arg Gly Pro Val Leu                        20     - #             25     - #             30                  - - TTA CAA AGC ACT TGG TTT TTG GAA AAG TTA GC - #G GCG TTT GAC AGA GAA          144                                                                       Leu Gln Ser Thr Trp Phe Leu Glu Lys Leu Al - #a Ala Phe Asp Arg Glu                    35         - #         40         - #         45                      - - AGG ATC CCT GAA AGG GTG GTG CAT GCT AAA GG - #A AGC GGG GCT TAT GGC          192                                                                       Arg Ile Pro Glu Arg Val Val His Ala Lys Gl - #y Ser Gly Ala Tyr Gly                50             - #     55             - #     60                          - - ACT TTC ACC GTG ACT AAA GAC ATC ACT AAA TA - #C ACT AAA GCG AAA ATT          240                                                                       Thr Phe Thr Val Thr Lys Asp Ile Thr Lys Ty - #r Thr Lys Ala Lys Ile            65                 - # 70                 - # 75                 - # 80       - - TTC TCT AAA GTG GGC AAA AAA ACA GAA TGC TT - #C TTC AGA TTT TCT ACT          288                                                                       Phe Ser Lys Val Gly Lys Lys Thr Glu Cys Ph - #e Phe Arg Phe Ser Thr                            85 - #                 90 - #                 95              - - GTG GCT GGT GAA AGA GGC AGT GCG GAT GCG GT - #A AGA GAC CCT AGA GGT          336                                                                       Val Ala Gly Glu Arg Gly Ser Ala Asp Ala Va - #l Arg Asp Pro Arg Gly                       100      - #           105      - #           110                  - - TTT GCG ATG AAG TAT TAC ACT GAA GAA GGT AA - #C TGG GAT TTA GTG GGG          384                                                                       Phe Ala Met Lys Tyr Tyr Thr Glu Glu Gly As - #n Trp Asp Leu Val Gly                   115          - #       120          - #       125                      - - AAC AAC ACG CCT GTT TTC TTT ATC CGT GAT GC - #G ATC AAA TTC CCT GAT          432                                                                       Asn Asn Thr Pro Val Phe Phe Ile Arg Asp Al - #a Ile Lys Phe Pro Asp               130              - #   135              - #   140                          - - TTC ATC CAC ACT CAA AAA CGA GAT CCT CAA AC - #C AAT TTG CCT AAC CAT          480                                                                       Phe Ile His Thr Gln Lys Arg Asp Pro Gln Th - #r Asn Leu Pro Asn His           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GAC ATG GTA TGG GAT TTT TGG AGC AAT GTT CC - #T GAA AGC TTA TAC        CAA      528                                                                    Asp Met Val Trp Asp Phe Trp Ser Asn Val Pr - #o Glu Ser Leu Tyr Gln                          165  - #               170  - #               175              - - GTA ACA TGG GTT ATG AGC GAT AGA GGT ATC CC - #T AAA TCT TTC CGC CAC          576                                                                       Val Thr Trp Val Met Ser Asp Arg Gly Ile Pr - #o Lys Ser Phe Arg His                       180      - #           185      - #           190                  - - ATG GAT GGT TTT GGC AGC CAC ACT TTC AGT CT - #T ATC AAC GCT AAA GGC          624                                                                       Met Asp Gly Phe Gly Ser His Thr Phe Ser Le - #u Ile Asn Ala Lys Gly                   195          - #       200          - #       205                      - - GAA CGC TTT TGG GTG AAA TTC CAC TTT GAA AC - #C ATG CAA GGC GTT AAG          672                                                                       Glu Arg Phe Trp Val Lys Phe His Phe Glu Th - #r Met Gln Gly Val Lys               210              - #   215              - #   220                          - - CAC TTG ACT AAC GAA GAA GCC GCA GAA ATC AG - #A AAG CAT GAT CCC GAT          720                                                                       His Leu Thr Asn Glu Glu Ala Ala Glu Ile Ar - #g Lys His Asp Pro Asp           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TCC AAT CAA AGG GAT TTA TTC AAT GCG ATC GC - #T AGA GGG GAT TTC        CCA      768                                                                    Ser Asn Gln Arg Asp Leu Phe Asn Ala Ile Al - #a Arg Gly Asp Phe Pro                          245  - #               250  - #               255              - - AAA TGG AAA TTA AGC ATT CAA GTG ATG CCA GA - #A GAG GAC GCT AAG AAG          816                                                                       Lys Trp Lys Leu Ser Ile Gln Val Met Pro Gl - #u Glu Asp Ala Lys Lys                       260      - #           265      - #           270                  - - TAT CGA TTC CAT CCG TTT GAT GTT ACT AAA AT - #T TGG TAT CTC CAA GAT          864                                                                       Tyr Arg Phe His Pro Phe Asp Val Thr Lys Il - #e Trp Tyr Leu Gln Asp                   275          - #       280          - #       285                      - - TAT CCA TTG ATG GAA GTG GGC ATT GTA GAG TT - #G AAT AAA AAT CCC GAA          912                                                                       Tyr Pro Leu Met Glu Val Gly Ile Val Glu Le - #u Asn Lys Asn Pro Glu               290              - #   295              - #   300                          - - AAC TAT TTC GCA GAA GTG GAG CAA GCG GCA TT - #C AGT CCG GCT AAT GTC          960                                                                       Asn Tyr Phe Ala Glu Val Glu Gln Ala Ala Ph - #e Ser Pro Ala Asn Val           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - GTT CCT GGA ATT GGC TAT AGC CCT GAT AGG AT - #G TTA CAA GGG CGC        TTG     1008                                                                    Val Pro Gly Ile Gly Tyr Ser Pro Asp Arg Me - #t Leu Gln Gly Arg Leu                          325  - #               330  - #               335              - - TTC TCT TAT GGA GAC ACA CAC CGC TAC CGC TT - #A GGG GTT AAT TAT CCT         1056                                                                       Phe Ser Tyr Gly Asp Thr His Arg Tyr Arg Le - #u Gly Val Asn Tyr Pro                       340      - #           345      - #           350                  - - CAA ATA CCG GTT AAT AAA CCA AGA TGC CCG TT - #C CAC TCT TCT AGC AGA         1104                                                                       Gln Ile Pro Val Asn Lys Pro Arg Cys Pro Ph - #e His Ser Ser Ser Arg                   355          - #       360          - #       365                      - - GAT GGT TAC ATG CAA AAC GGG TAT TAC GGC TC - #T TTA CAA AAC TAT ACG         1152                                                                       Asp Gly Tyr Met Gln Asn Gly Tyr Tyr Gly Se - #r Leu Gln Asn Tyr Thr               370              - #   375              - #   380                          - - CCT AGC TCA TTG CCT GGC TAT AAA GAA GAT AA - #G AGT GCA AGG GAT CCT         1200                                                                       Pro Ser Ser Leu Pro Gly Tyr Lys Glu Asp Ly - #s Ser Ala Arg Asp Pro           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - AAG TTC AAC TTA GCT CAT ATT GAG AAA GAG TT - #T GAA GTG TGG AAT        TGG     1248                                                                    Lys Phe Asn Leu Ala His Ile Glu Lys Glu Ph - #e Glu Val Trp Asn Trp                          405  - #               410  - #               415              - - GAT TAC AGA GCT GAG GAT AGC GAT TAC TAC AC - #C CAA CCA GGT GAT TAC         1296                                                                       Asp Tyr Arg Ala Glu Asp Ser Asp Tyr Tyr Th - #r Gln Pro Gly Asp Tyr                       420      - #           425      - #           430                  - - TAC CGC TCA TTG CCA GCT GAT GAA AAA GAA AG - #G TTG CAT GAC ACT ATT         1344                                                                       Tyr Arg Ser Leu Pro Ala Asp Glu Lys Glu Ar - #g Leu His Asp Thr Ile                   435          - #       440          - #       445                      - - GGA GAG TCT TTA GCT CAT GTT ACC CAT AAG GA - #A ATT GTG GAT AAA CAA         1392                                                                       Gly Glu Ser Leu Ala His Val Thr His Lys Gl - #u Ile Val Asp Lys Gln               450              - #   455              - #   460                          - - TTG GAG CAT TTC AAG AAA GCT GAC CCC AAA TA - #C GCT GAG GGA GTT AAA         1440                                                                       Leu Glu His Phe Lys Lys Ala Asp Pro Lys Ty - #r Ala Glu Gly Val Lys           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - AAA GCT CTT GAA AAA CAC CAA AAA ATG ATG AA - #A GAC ATG CAT GGA        AAA     1488                                                                    Lys Ala Leu Glu Lys His Gln Lys Met Met Ly - #s Asp Met His Gly Lys                          485  - #               490  - #               495              - - GAC ATG CAC CAC ACG AAA AAG AAA AAG TAA  - #                  - #             1518                                                                     Asp Met His His Thr Lys Lys Lys Lys                                                       500      - #           505                                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 505 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Val Asn Lys Asp Val Lys Gln Thr Thr Al - #a Phe Gly Thr Pro Val        1               5 - #                 10 - #                 15              - - Trp Asp Asp Asn Asn Val Ile Thr Ala Gly Pr - #o Arg Gly Pro Val Leu                   20     - #             25     - #             30                  - - Leu Gln Ser Thr Trp Phe Leu Glu Lys Leu Al - #a Ala Phe Asp Arg Glu               35         - #         40         - #         45                      - - Arg Ile Pro Glu Arg Val Val His Ala Lys Gl - #y Ser Gly Ala Tyr Gly           50             - #     55             - #     60                          - - Thr Phe Thr Val Thr Lys Asp Ile Thr Lys Ty - #r Thr Lys Ala Lys Ile       65                 - # 70                 - # 75                 - # 80       - - Phe Ser Lys Val Gly Lys Lys Thr Glu Cys Ph - #e Phe Arg Phe Ser Thr                       85 - #                 90 - #                 95              - - Val Ala Gly Glu Arg Gly Ser Ala Asp Ala Va - #l Arg Asp Pro Arg Gly                  100      - #           105      - #           110                  - - Phe Ala Met Lys Tyr Tyr Thr Glu Glu Gly As - #n Trp Asp Leu Val Gly              115          - #       120          - #       125                      - - Asn Asn Thr Pro Val Phe Phe Ile Arg Asp Al - #a Ile Lys Phe Pro Asp          130              - #   135              - #   140                          - - Phe Ile His Thr Gln Lys Arg Asp Pro Gln Th - #r Asn Leu Pro Asn His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Met Val Trp Asp Phe Trp Ser Asn Val Pr - #o Glu Ser Leu Tyr        Gln                                                                                             165  - #               170  - #               175             - - Val Thr Trp Val Met Ser Asp Arg Gly Ile Pr - #o Lys Ser Phe Arg His                  180      - #           185      - #           190                  - - Met Asp Gly Phe Gly Ser His Thr Phe Ser Le - #u Ile Asn Ala Lys Gly              195          - #       200          - #       205                      - - Glu Arg Phe Trp Val Lys Phe His Phe Glu Th - #r Met Gln Gly Val Lys          210              - #   215              - #   220                          - - His Leu Thr Asn Glu Glu Ala Ala Glu Ile Ar - #g Lys His Asp Pro Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Asn Gln Arg Asp Leu Phe Asn Ala Ile Al - #a Arg Gly Asp Phe        Pro                                                                                             245  - #               250  - #               255             - - Lys Trp Lys Leu Ser Ile Gln Val Met Pro Gl - #u Glu Asp Ala Lys Lys                  260      - #           265      - #           270                  - - Tyr Arg Phe His Pro Phe Asp Val Thr Lys Il - #e Trp Tyr Leu Gln Asp              275          - #       280          - #       285                      - - Tyr Pro Leu Met Glu Val Gly Ile Val Glu Le - #u Asn Lys Asn Pro Glu          290              - #   295              - #   300                          - - Asn Tyr Phe Ala Glu Val Glu Gln Ala Ala Ph - #e Ser Pro Ala Asn Val      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Pro Gly Ile Gly Tyr Ser Pro Asp Arg Me - #t Leu Gln Gly Arg        Leu                                                                                             325  - #               330  - #               335             - - Phe Ser Tyr Gly Asp Thr His Arg Tyr Arg Le - #u Gly Val Asn Tyr Pro                  340      - #           345      - #           350                  - - Gln Ile Pro Val Asn Lys Pro Arg Cys Pro Ph - #e His Ser Ser Ser Arg              355          - #       360          - #       365                      - - Asp Gly Tyr Met Gln Asn Gly Tyr Tyr Gly Se - #r Leu Gln Asn Tyr Thr          370              - #   375              - #   380                          - - Pro Ser Ser Leu Pro Gly Tyr Lys Glu Asp Ly - #s Ser Ala Arg Asp Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Lys Phe Asn Leu Ala His Ile Glu Lys Glu Ph - #e Glu Val Trp Asn        Trp                                                                                             405  - #               410  - #               415             - - Asp Tyr Arg Ala Glu Asp Ser Asp Tyr Tyr Th - #r Gln Pro Gly Asp Tyr                  420      - #           425      - #           430                  - - Tyr Arg Ser Leu Pro Ala Asp Glu Lys Glu Ar - #g Leu His Asp Thr Ile              435          - #       440          - #       445                      - - Gly Glu Ser Leu Ala His Val Thr His Lys Gl - #u Ile Val Asp Lys Gln          450              - #   455              - #   460                          - - Leu Glu His Phe Lys Lys Ala Asp Pro Lys Ty - #r Ala Glu Gly Val Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Lys Ala Leu Glu Lys His Gln Lys Met Met Ly - #s Asp Met His Gly        Lys                                                                                             485  - #               490  - #               495             - - Asp Met His His Thr Lys Lys Lys Lys                                                  500      - #           505                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1518 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Heliobacter - #pylori                                           (B) STRAIN: RU1                                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1515                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 1..1515                                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3:                        - - ATG GTT AAT AAA GAT GTG AAA CAA ACC ACT GC - #T TTT GGC GCT CCC GTT           48                                                                       Met Val Asn Lys Asp Val Lys Gln Thr Thr Al - #a Phe Gly Ala Pro Val             1               5 - #                 10 - #                 15              - - TGG GAT GAT AAC AAT GTG ATT ACG GCT GGT CC - #T AGA GGT CCT GTT TTA           96                                                                       Trp Asp Asp Asn Asn Val Ile Thr Ala Gly Pr - #o Arg Gly Pro Val Leu                        20     - #             25     - #             30                  - - TTA CAA AGC ACT TGG TTT TTG GAA AAG TTA GC - #A GCG TTT GAC AGA GAA          144                                                                       Leu Gln Ser Thr Trp Phe Leu Glu Lys Leu Al - #a Ala Phe Asp Arg Glu                    35         - #         40         - #         45                      - - AGG ATC CCT GAA AGG GTA GTG CAT GCT AAA GG - #A AGC GGG GCT TAT GGC          192                                                                       Arg Ile Pro Glu Arg Val Val His Ala Lys Gl - #y Ser Gly Ala Tyr Gly                50             - #     55             - #     60                          - - ACT TTC ACC GTG ACT AAA GAC ATC ACT AAA TA - #C ACT AAA GCG AAG ATT          240                                                                       Thr Phe Thr Val Thr Lys Asp Ile Thr Lys Ty - #r Thr Lys Ala Lys Ile            65                 - # 70                 - # 75                 - # 80       - - TTC TCT AAA GTG GGC AAA AAA ACC GAA TGC TT - #T TTC AGG TTT TCT ACT          288                                                                       Phe Ser Lys Val Gly Lys Lys Thr Glu Cys Ph - #e Phe Arg Phe Ser Thr                            85 - #                 90 - #                 95              - - GTG GCT GGT GAA AGA GGC AGT GCG GAT GCA GT - #G AGA GAC CCT AGA GGT          336                                                                       Val Ala Gly Glu Arg Gly Ser Ala Asp Ala Va - #l Arg Asp Pro Arg Gly                       100      - #           105      - #           110                  - - TTT GCG ATG AAG TAT TAC ACT GAA GAA GGT AA - #C TGG GAT TTA GTA GGG          384                                                                       Phe Ala Met Lys Tyr Tyr Thr Glu Glu Gly As - #n Trp Asp Leu Val Gly                   115          - #       120          - #       125                      - - AAC AAC ACG CCT GTT TTC TTT ATC CGT GAT GC - #G ATC AAA TTC CCT GAT          432                                                                       Asn Asn Thr Pro Val Phe Phe Ile Arg Asp Al - #a Ile Lys Phe Pro Asp               130              - #   135              - #   140                          - - TTC ATC CAC ACC CAA AAA AGA GAC CCT CAA AC - #C AAT TTG CCT AAC CAC          480                                                                       Phe Ile His Thr Gln Lys Arg Asp Pro Gln Th - #r Asn Leu Pro Asn His           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GAC ATG GTA TGG GAT TTT TGG AGT AAT GTT CC - #T GAA AGC TTG TAT        CAA      528                                                                    Asp Met Val Trp Asp Phe Trp Ser Asn Val Pr - #o Glu Ser Leu Tyr Gln                          165  - #               170  - #               175              - - GTA ACA TGG GTT ATG AGC GAT AGA GGG ATC CC - #T AAA TCT TTC CGC CAC          576                                                                       Val Thr Trp Val Met Ser Asp Arg Gly Ile Pr - #o Lys Ser Phe Arg His                       180      - #           185      - #           190                  - - ATG GAT GGT TTT GGC AGC CAC ACT TTC AGT CT - #T ATC AAC GCT AAG GGC          624                                                                       Met Asp Gly Phe Gly Ser His Thr Phe Ser Le - #u Ile Asn Ala Lys Gly                   195          - #       200          - #       205                      - - GAA CGC TTT TGG GTG AAA TTC CAC TTT CAC AC - #C ATG CAA GGC GTT AAG          672                                                                       Glu Arg Phe Trp Val Lys Phe His Phe His Th - #r Met Gln Gly Val Lys               210              - #   215              - #   220                          - - CAC TTG ACT AAC GAA GAA GCC GCA GAA GTC AG - #A AAA TAT GAT CCT GAT          720                                                                       His Leu Thr Asn Glu Glu Ala Ala Glu Val Ar - #g Lys Tyr Asp Pro Asp           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - TCC AAT CAA AGG GAT TTA TTC AAT GCG ATC GC - #T AGA GGG GAT TTC        CCA      768                                                                    Ser Asn Gln Arg Asp Leu Phe Asn Ala Ile Al - #a Arg Gly Asp Phe Pro                          245  - #               250  - #               255              - - AAA TGG AAA TTA AGC ATT CAA GTG ATG CCA GA - #A GAA GAT GCT AAG AAG          816                                                                       Lys Trp Lys Leu Ser Ile Gln Val Met Pro Gl - #u Glu Asp Ala Lys Lys                       260      - #           265      - #           270                  - - TAT CGA TTC CAT CCG TTT GAT GTT ACT AAA AT - #T TGG TAT CTC CAA GAT          864                                                                       Tyr Arg Phe His Pro Phe Asp Val Thr Lys Il - #e Trp Tyr Leu Gln Asp                   275          - #       280          - #       285                      - - TAT CCG TTG ATG GAA GTG GGC ATT GTA GAG TT - #G AAT AAA AAT CCA GAA          912                                                                       Tyr Pro Leu Met Glu Val Gly Ile Val Glu Le - #u Asn Lys Asn Pro Glu               290              - #   295              - #   300                          - - AAC TAT TTT GCA GAA GTG GAG CAA GTG GCA TT - #C ACT CCG GCT AAT GTC          960                                                                       Asn Tyr Phe Ala Glu Val Glu Gln Val Ala Ph - #e Thr Pro Ala Asn Val           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - GTT CCT GGA ATT GGC TAT AGC CCT GAT AGG AT - #G TTA CAA GGA CGC        TTG     1008                                                                    Val Pro Gly Ile Gly Tyr Ser Pro Asp Arg Me - #t Leu Gln Gly Arg Leu                          325  - #               330  - #               335              - - TTC TCT TAT GGG GAC ACA CAC CGC TAC CGC TT - #A GGG GTT AAT TAT CCT         1056                                                                       Phe Ser Tyr Gly Asp Thr His Arg Tyr Arg Le - #u Gly Val Asn Tyr Pro                       340      - #           345      - #           350                  - - CAA ATA CCG GTT AAT AAA CCA AGA TGC CCG TT - #C CAC TCT TCT AGC AGA         1104                                                                       Gln Ile Pro Val Asn Lys Pro Arg Cys Pro Ph - #e His Ser Ser Ser Arg                   355          - #       360          - #       365                      - - GAT GGT TAC ATG CAA AAC GGA TAC TAC GGC TC - #T TTA CAA AAC TAT ACG         1152                                                                       Asp Gly Tyr Met Gln Asn Gly Tyr Tyr Gly Se - #r Leu Gln Asn Tyr Thr               370              - #   375              - #   380                          - - CCT AGC TCA TTG CCA GGT TAT AAA GAA GAT AA - #G AGC GCG AGA GAT CCT         1200                                                                       Pro Ser Ser Leu Pro Gly Tyr Lys Glu Asp Ly - #s Ser Ala Arg Asp Pro           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - AAG TTC AAC TTA GCT CAT ATT GAG AAA GAG TT - #T GAA GTG TGG AAT        TGG     1248                                                                    Lys Phe Asn Leu Ala His Ile Glu Lys Glu Ph - #e Glu Val Trp Asn Trp                          405  - #               410  - #               415              - - GAT TAC AGG GCT GAT GAT AGC GAT TAC TAC AC - #C CAA CCA GGT GAT TAC         1296                                                                       Asp Tyr Arg Ala Asp Asp Ser Asp Tyr Tyr Th - #r Gln Pro Gly Asp Tyr                       420      - #           425      - #           430                  - - TAC CGC TCA TTG CCA GCT GAT GAA AAA GAA AG - #G TTG CAT GAC ACT ATT         1344                                                                       Tyr Arg Ser Leu Pro Ala Asp Glu Lys Glu Ar - #g Leu His Asp Thr Ile                   435          - #       440          - #       445                      - - GGA GAG TCT TTG GCT CAT GTT ACC CAT AAG GA - #A ATT GTG GAT AAA CAA         1392                                                                       Gly Glu Ser Leu Ala His Val Thr His Lys Gl - #u Ile Val Asp Lys Gln               450              - #   455              - #   460                          - - TTG GAG CAT TTC AAG AAA GCT GAT CCC AAA TA - #C GCT GAG GGA GTT AAA         1440                                                                       Leu Glu His Phe Lys Lys Ala Asp Pro Lys Ty - #r Ala Glu Gly Val Lys           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - AAA GCT CTT GAA AAA CAC CAA AAG ATG ATG AA - #A GAC ATG CAT GGA        AAA     1488                                                                    Lys Ala Leu Glu Lys His Gln Lys Met Met Ly - #s Asp Met His Gly Lys                          485  - #               490  - #               495              - - GAC ATG CAC CAC ACA AAA AAG AAA AAG TAA  - #                  - #             1518                                                                     Asp Met His His Thr Lys Lys Lys Lys                                                       500      - #           505                                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 505 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Val Asn Lys Asp Val Lys Gln Thr Thr Al - #a Phe Gly Ala Pro Val        1               5 - #                 10 - #                 15              - - Trp Asp Asp Asn Asn Val Ile Thr Ala Gly Pr - #o Arg Gly Pro Val Leu                   20     - #             25     - #             30                  - - Leu Gln Ser Thr Trp Phe Leu Glu Lys Leu Al - #a Ala Phe Asp Arg Glu               35         - #         40         - #         45                      - - Arg Ile Pro Glu Arg Val Val His Ala Lys Gl - #y Ser Gly Ala Tyr Gly           50             - #     55             - #     60                          - - Thr Phe Thr Val Thr Lys Asp Ile Thr Lys Ty - #r Thr Lys Ala Lys Ile       65                 - # 70                 - # 75                 - # 80       - - Phe Ser Lys Val Gly Lys Lys Thr Glu Cys Ph - #e Phe Arg Phe Ser Thr                       85 - #                 90 - #                 95              - - Val Ala Gly Glu Arg Gly Ser Ala Asp Ala Va - #l Arg Asp Pro Arg Gly                  100      - #           105      - #           110                  - - Phe Ala Met Lys Tyr Tyr Thr Glu Glu Gly As - #n Trp Asp Leu Val Gly              115          - #       120          - #       125                      - - Asn Asn Thr Pro Val Phe Phe Ile Arg Asp Al - #a Ile Lys Phe Pro Asp          130              - #   135              - #   140                          - - Phe Ile His Thr Gln Lys Arg Asp Pro Gln Th - #r Asn Leu Pro Asn His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Met Val Trp Asp Phe Trp Ser Asn Val Pr - #o Glu Ser Leu Tyr        Gln                                                                                             165  - #               170  - #               175             - - Val Thr Trp Val Met Ser Asp Arg Gly Ile Pr - #o Lys Ser Phe Arg His                  180      - #           185      - #           190                  - - Met Asp Gly Phe Gly Ser His Thr Phe Ser Le - #u Ile Asn Ala Lys Gly              195          - #       200          - #       205                      - - Glu Arg Phe Trp Val Lys Phe His Phe His Th - #r Met Gln Gly Val Lys          210              - #   215              - #   220                          - - His Leu Thr Asn Glu Glu Ala Ala Glu Val Ar - #g Lys Tyr Asp Pro Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Asn Gln Arg Asp Leu Phe Asn Ala Ile Al - #a Arg Gly Asp Phe        Pro                                                                                             245  - #               250  - #               255             - - Lys Trp Lys Leu Ser Ile Gln Val Met Pro Gl - #u Glu Asp Ala Lys Lys                  260      - #           265      - #           270                  - - Tyr Arg Phe His Pro Phe Asp Val Thr Lys Il - #e Trp Tyr Leu Gln Asp              275          - #       280          - #       285                      - - Tyr Pro Leu Met Glu Val Gly Ile Val Glu Le - #u Asn Lys Asn Pro Glu          290              - #   295              - #   300                          - - Asn Tyr Phe Ala Glu Val Glu Gln Val Ala Ph - #e Thr Pro Ala Asn Val      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Pro Gly Ile Gly Tyr Ser Pro Asp Arg Me - #t Leu Gln Gly Arg        Leu                                                                                             325  - #               330  - #               335             - - Phe Ser Tyr Gly Asp Thr His Arg Tyr Arg Le - #u Gly Val Asn Tyr Pro                  340      - #           345      - #           350                  - - Gln Ile Pro Val Asn Lys Pro Arg Cys Pro Ph - #e His Ser Ser Ser Arg              355          - #       360          - #       365                      - - Asp Gly Tyr Met Gln Asn Gly Tyr Tyr Gly Se - #r Leu Gln Asn Tyr Thr          370              - #   375              - #   380                          - - Pro Ser Ser Leu Pro Gly Tyr Lys Glu Asp Ly - #s Ser Ala Arg Asp Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Lys Phe Asn Leu Ala His Ile Glu Lys Glu Ph - #e Glu Val Trp Asn        Trp                                                                                             405  - #               410  - #               415             - - Asp Tyr Arg Ala Asp Asp Ser Asp Tyr Tyr Th - #r Gln Pro Gly Asp Tyr                  420      - #           425      - #           430                  - - Tyr Arg Ser Leu Pro Ala Asp Glu Lys Glu Ar - #g Leu His Asp Thr Ile              435          - #       440          - #       445                      - - Gly Glu Ser Leu Ala His Val Thr His Lys Gl - #u Ile Val Asp Lys Gln          450              - #   455              - #   460                          - - Leu Glu His Phe Lys Lys Ala Asp Pro Lys Ty - #r Ala Glu Gly Val Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Lys Ala Leu Glu Lys His Gln Lys Met Met Ly - #s Asp Met His Gly        Lys                                                                                             485  - #               490  - #               495             - - Asp Met His His Thr Lys Lys Lys Lys                                                  500      - #           505                                         - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Lys Lys Ile Val Phe Lys Glu Tyr Val                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Val Asn Lys Asp Val Lys Gln Thr Thr Al - #a Phe Gly Thr Pro          1               5   - #                10  - #                15             __________________________________________________________________________

We claim:
 1. A vaccine composition for use in the treatment orprevention of Helicobacter infection in a mammalian host, which vaccineconsists essentially of an immunologically effective amount ofHelicobacter pylori catalase, together with a mucosal adjuvant and apharmaceutically acceptable carrier or diluent.
 2. A compositionaccording to claim 1, wherein the catalase is recombinant catalase.
 3. Amethod for the treatment or prevention of Helicobacter infection in amammalian host, which comprises administration to said host of animmunologically effective amount of an antigenic preparation consistingessentially of Helicobacter pylori catalase, together with a mucosaladjuvant and a pharmaceutically acceptable carrier or diluent.
 4. Amethod according to claim 3, wherein said antigenic preparation isorally administered to said host.
 5. A method according to claim 3,wherein said host is a human.
 6. A method according to claim 3, whereinthe catalase is recombinant catalase.
 7. A method of producing avaccine, comprising the step of bringing an antigenic preparationconsisting essentially of Helicobacter pylori catalase, together with amucosal adjuvant and a pharmaceutically acceptable carrier or diluent,into a form suitable for administration to a mammal.
 8. A vaccinecomposition for use in the treatment or prevention of Helicobacterinfection in a mammalian host, consisting essentially of animmunologically effective amount of Helicobacter pylori catalase and anadditional Helicobacter antigen, together with a mucosal adjuvant and apharmaceutically acceptable carrier or diluent, wherein said additionalantigen is Helicobacter urease or Helicobacter lipopolysaccharide.
 9. Avaccine composition for use in the treatment or prevention ofHelicobacter infection in a mammalian host, consisting essentially of animmunologically effective amount of Helicobacter pylori catalase,together with Helicobacter urease and Helicobacter lipopolysaccharide,and further together with a mucosal adjuvant and a pharmaceuticallyacceptable carrier or diluent.
 10. A method for the treatment orprevention of Helicobacter infection in a mammalian host, whichcomprises administration to said host of an immunologically effectiveamount of an antigenic preparation consisting essentially ofHelicobacter pylori catalase and an additional Helicobacter antigen,together with a mucosal adjuvant and a pharmaceutically acceptablecarrier or diluent, wherein said additional antigen is Helicobacterurease or Helicobacter lipopolysaccharide.
 11. A method for thetreatment or prevention of Helicobacter infection in a mammalian host,which comprises administration to said host of an immunologicallyeffective amount of an antigenic preparation consisting essentially ofHelicobacter pylori catalase, together with Helicobacter urease andHelicobacter lipopolysaccharide, and further together with a mucosaladjuvant and a pharmaceutically acceptable carrier or diluent.
 12. Amethod of producing a vaccine, comprising the step of bringing anantigenic preparation into a form suitable for administration to amammal, wherein said preparation consists essentially of Helicobacterpylori catalase and an additional Helicobacter antigen, together with amucosal adjuvant and a pharmaceutically acceptable carrier or diluent,and wherein said additional antigen is Helicobacter urease orHelicobacter lipopolysaccharide.
 13. Method of producing a vaccine,comprising the step of bringing an antigenic preparation into a formsuitable for administration to a mammal, wherein said preparationconsists essentially of Helicobacter pylori catalase, and Helicobacterurease and Helicobacter lipopolysaccharide, together with a mucosaladjuvant and a pharmaceutically acceptable carrier or diluent.